Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3' end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.

中文翻译：

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通过引入用于蛋白质结构和功能研究的沉默限制性位点，在基于PCR的多步定向质粒诱变的一步法中增强突变体筛选。

定点诱变（SDM）已被广泛用于研究蛋白质的结构和功能。一种基于一步聚合酶链反应（PCR）的多点定向质粒诱变方法，在引物3'端具有扩展的非重叠序列，可提高PCR扩增效率和多点诱变能力。在这里，我们通过利用SDM-Assist软件生成幽门螺杆菌嗜中性粒细胞激活蛋白（HP-NAP）的突变体，在该基于PCR的SDM方法中使用的引物中引入了沉默限制性位点，该突变体的基因GC含量较低。HP-NAP突变体是通过这种改良的诱变方法高效生成的，由于存在沉默限制性酶切位点，因此可以通过简单的限制性酶切酶快速鉴定出来。